The EGFR Receptor Family
The epidermal growth factor receptor (EGFR) family (also known as the ErbB family) is a subgroup of the receptor tyrosine kinases (RTKs) and consists of four members: HER1/EGFR/ErbB, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The members of the EGFR family are closely related single-chain modular glycoproteins with an extracellular ligand binding region, a single transmembrane domain and an intracellular tyrosine kinase (reviewed in Ferguson (2008) Annu Rev Biophys. 37: 353-373). In normal physiological settings the ErbB family regulates key events in coordination of cell growth, differentiation and migration (Citri et al. (2006) Nat Rev Mol Cell Biol. 7: 505-516). EGFR, HER2 and HER3 are believed to play crucial roles in the malignant transformation of normal cells and in the continued growth of cancer cells (pro-survival pathway). EGFR and HER2 have been found to be overexpressed by many epithelial cancers (Slamon et al. (1987) Science, 235: 177-182; Arteaga (2002) Oncologist 7 Suppl 4: 31-39; Bodey et al. (1997) Anticancer Res. 17: 1319-1330; Rajkumar et al. (1996) J. Pathol. 179: 381-385). Overexpression of EGFR and HER2 has furthermore been linked to disease progression, reduced survival, poor response and chemotherapy resistance in several human epithelial cancers (Slamon et al. (1987) supra; Baselga et al. (2002) Oncologist 7 Suppl 4: 2-8).
HER3 Structure
The third member of the ErbB family, known as human epidermal growth factor receptor 3 (HER3, ErbB3) was identified in 1989 by Kraus et al. (Proc Natl Acad Sci USA 1989; 86: 9193-9197). The HER3 gene encodes a protein of 1342 amino acids with striking structural similarities to EGFR and HER2. Features such as overall size, four extracellular subdomains (I-IV) with two cysteine clusters (domains II and IV), and a tyrosine kinase domain show structural similarities to EGFR and HER2 (Cho and Leahy (2002) Science, 297: 1330-1333). The tyrosine kinase domain of HER3 shows 59% sequence homology to the tyrosine kinase domain of EGFR (Brennan et al. (2000) Oncogene, 19: 6093-6101).
Regulation of HER3 Activation
Neu differentiation factor (NDF), heregulin (HRG) and neuregulin 1 (NRG1) are synonyms for the glycoprotein which is a ligand for HER3 (Peles et al. (1992) Cell, 69: 205-216; Wen et al. (1992) Cell, 69: 559-572). At least 15 isoforms of the NRG1 protein have been identified. The isoforms are produced from the single NRG1 gene through alternative splicing and multiple promoters (Falls et al. (2003) Exp Cell Res, 284: 14-30). Three structural characteristics apply for the functional differences of the isoforms. These structural characteristics are the type of EGF-like domain (α or β), the N-terminal sequence (type I, II or III) and whether the isoform is initially synthesized as a transmembrane or non-membrane protein (Falls et al. (2003) supra). The type I sub-group of NRG1 isoforms have a unique N-terminal sequence followed by an immunoglobulin-like domain and then an EGF-like domain. Type II variants contain an N-terminal kringle-like sequence, the immunoglobulin domain and the EGF-like domain. The type III variants contain an N-terminal hydrophobic domain within a cysteine-rich region, omit the immunoglobulin domain and then continue into the EGF-like domain and various downstream alternative exons. Downstream from the EGF-like domain the NRG1 isoform may contain a linker sequence, a transmembrane domain and a cytoplasmic tail (Falls et al. (2003) supra). Some of the NRG1 isoforms are subject to glycosylation in the spacer region between the immunoglobulin-like domain and the EGF-like domain (Hayes et al. (2008) J Mammary Gland Biol Neoplasia, 13: 205-214).
As is the case for EGFR, HER3 exists in a tethered conformation and in an extended conformation. In the tethered conformation the dimerization arm is buried by interactions with domain IV, leaving domains I and III too far apart for efficient ligand binding (Cho and Leahy et al. (2002) supra). Ligand binding to the extracellular domains I and III occurs in the extended conformation of HER3 and leads to heterodimerization with other members of the ErbB family (or other RTK members, e.g. MET), the extended and ligand-bound HER3 molecule preferentially heterodimerizing with HER2 (Pinkas-Kramarski et al. (1996) EMBO J, 15: 2452-2467). No HER3 homodimers are formed upon ligand binding (Ferguson et al. (2000) EMBO J, 19: 4632-4643).
In contrast to EGFR and HER2, the tyrosine kinase of HER3 has impaired catalytic activity, insufficient for any detectable biological response (Pinkas-Kramarski et al. (1996) supra; Guy et al. (1994) Proc Natl Aced Sci USA, 91: 8132-8136). Two amino acid residues which are highly conserved in the catalytic domains of protein kinases (Hanks et al. (1988) Science, 241: 42-52) are altered in the catalytic domain of HER3. These are the substitution of aspargine for aspartic acid at residue 815 and substitution of histamine for glutamate at residue 740. The two amino acid substitutions may be the reason why HER3 lacks catalytic activity of its tyrosine kinase domain (Plowman et al. (1990) Proc Natl Acad Sci USA, 87: 4905-4909). Because of the impaired intrinsic kinase activity of HER3, the receptor needs to heterodimerize with another ErbB family member in order to respond to its own ligand binding (Berger et al. (2004) FEBS Lett, 569: 332-336).
Termination of HER3 Signaling
Little is known about endocytosis of HER3. Moreover, different studies have suggested that HER3 is endocytosis impaired to the same extent as HER2 (Baulida et al. (1996) J Biol Chem, 271: 5251-5257). In agreement with this the HER3-NRG1 complex was found to be internalized less efficiently and slower than the EGFR-EGF complex, supporting that HER3 is not endocytosed as efficiently as EGFR (Baulida et al. (1997) Exp Cell Res, 232: 167-172; Waterman et al. (1999) EMBO J, 18: 3348-3358). However, when the C-terminal tail of EGFR was replaced with the C-terminal tail of HER3, EGFR became endocytosis impaired, suggesting that a region in the C-terminus of HER3 protects the receptor against internalization (Waterman et al. (1999) supra). It has also been suggested that NRG1 does not efficiently target HER3 to degradation due to the dissociation of the ligand-receptor complexes in endosomes, as it is observed when EGF is activated by TGFα (Waterman et al. (1999) supra).
Expression and Physiological Role of HER3
HER3 has like EGFR and HER2 been shown to be of importance in the mammary gland development (Schroeder et al. (1998) Cell Growth Differ, 9: 451-464). While EGFR and HER2 are highly expressed and co-localized in the pubscent mouse mammary gland, HER3 is only expressed at low levels in postpubscent mammary glands from virgin mice, but is expressed at higher levels during pregnancy and lactation (Schroeder et al. (1998) supra). The higher expression levels of HER3 during pregnancy and lactation implies the importance of HER3 in the later stages of mammary gland development and differentiation (Jackson-Fisher et al. (2008) Breast Cancer Res, 10: R96). Studies with HER3-deficient mice further indicated the regulatory role of HER3 in morphogenesis of mammary epithelium through the PI3K/AKT signaling pathway (Jackson-Fisher et al. (2008) supra). Other studies showed high levels of HER3 expression by ductal epithelial cells in rats by day 14-16 of pregnancy, also demonstrating the regulatory role of HER3 in morphogenesis of mammary epithelium (Darcy et al. (2000) J Histochem Cytochem, 48: 63-80).
Targeted knockout of the HER3 gene in mice resulted in embryonic lethality at day 13.5 due to underdeveloped cardiac valves which were unable to support proper cardiac function due to blood reflux (Erickson et al. (1997) Development, 124: 4999-5011). Other defects include abnormalities in brain development, especially in the midbrain region including the cerebellum, and severe defects in Schwann cells of peripheral axons of sensory and motor neurons (Erickson et al. (1997) supra; Riethmacher et al. (1997) Nature, 389: 725-730).
In vitro studies have also implicated HER3, in combination with HER2, in the development of keratinocytes (Marikovsky et al. (1995) Oncogene, 10: 1403-1411), Schwann cell precursors (Syroid et al. (1996) Proc Natl Aced Sci USA, 93: 9229-9234), oligodendrocytes (Vartanian et al. (1997) J Cell Biol, 137: 211-220) and the neuromuscular synapse (Zhu et al. (1995) EMBO J, 14: 5842-5848).
The tissue distribution of HER3 is not much different from EGFR (www.proteinatlas.org). Despite the impaired kinase activity of HER3, the receptor plays an essential role in the ErbB network through the PI3K/AKT signaling (Citri et al. (2003) Exp Cell Res, 284: 54-65). Due to the requirement of heterodimerization for initiation of signaling, the physiological role of HER3 may overall resemble those identified for EGFR and HER2. The precise role of HER3 in the human adults is unknown, however, due to the embryonic lethality of HER3 knockout in mice and the sparse data on HER3 inhibition.
HER3 and Cancer
HER3 is unique in its ability to channel ErbB signaling to the PI3K/AKT signaling pathway, which favors tumor growth and progression (Prigent et al. (1994) EMBO J, 13: 2831-2841). The critical role of HER3 in regulation of tumor growth is also supported by the observation that HER2 overexpression in human breast cancer often is associated with higher levels of HER3 expression (Naidu et al. (1998) Br J Cancer, 78: 1385-1390). Moreover, overexpression of HRG results in increased transformation and tumorigenicity (Atlas et al. (2003) Mol Cancer Res, 1: 165-175), while blockade of NRG inhibits tumorigenicity and metastasis (Tsai et al. (2003) Oncogene, 22: 761-768), indicating the importance of the presence of a HER3 ligand for cancer development.
The presence of HER2 homodimers on the cell surface and thereby exaggeration of HER2 signaling causes transformation of epithelial cells (reviewed in Yarden and Sliwkowski (2001) Nat Rev Mol Cell Biol, 2: 127-137). However the HER2-HER3 dimer has the ability to induce signal transduction through both the mitogen-activated protein kinase (MAPK) and the AKT pathway. Activation of both the MAPK pathway and the AKT pathway implies the additional oncogenic potential of the HER2-HER3 heterodimer compared to the HER2 homodimer (reviewed in Citri et al. (2003) supra).
High expression of HER3 is found in many of the same tumor types that overexpress HER2, including bladder and colorectal cancer in addition to breast cancer (Bodey et al. (1997) Anticancer Res, 17: 1319-1330; Rajkumar et al. (1996) J Pathol, 179: 381-385; Lemoine et al. (1992) BrJ Cancer, 66: 1116-1121; Maurer et al. (1998) Hum Pathol, 29: 771-777). While more studies are needed to establish the association between HER3 overexpression and clinical outcome, the clinical indications support the results from in vitro studies that neither HER2 nor HER3 can be considered as stand-alone receptors in relation to cancer.
Anti-HER3 Antibodies
A number of anti-HER3 antibodies have been described in the literature. See, for example, WO 2011/060206, WO 2011/044311, WO 2011/022727, WO 2010/127181, WO 2008/100624, WO 2007/077028, WO 03/013602 and WO 97/35885.
AMG 888 (Amgen/Daiichi Sankyo) is a fully human monoclonal antibody that is said to inhibit human HER3 oncogenic signaling. AMG 888 is currently being investigated in clinical trials for treatment of cancer.
MM-121 (Merrimack Pharmaceuticals) is an anti-HER3 antibody that is said to block heregulin binding to and hence activation of HER3; see WO 2010/019952 and Schoeberl et al., Cancer Res. 70(6):2485-94, March 2010. MM-121 is also currently being investigated in clinical trials for treatment of cancer.
Pertuzumab is an anti-HER2 antibody that functions as a HER dimerization inhibitor which inhibits dimerization of HER2 to HER3 and the other EGFR receptors. Franklin et al. (Cancer Cell 2004, 5(4):317-28) disclose that pertuzumab binds HER2 near the center of domain II, sterically blocking a binding pocket necessary for HER2-HER3 heterodimerization and signaling. The amino acid sequence of pertuzumab is disclosed in WO 2006/033700 and US 2006/0121044 A1.
In spite of the fact that certain anti-HER3 antibodies are known and in some cases being investigated in clinical trials, no anti-HER3 antibodies are currently approved for therapeutic use. In view of the critical role of HER3 in regulation of tumor growth as outlined above, there is therefore a need for new antibodies that target the HER3 receptor as well as mixtures of such anti-HER3 antibodies.